Gene therapy has applications in the treatment of infectious, vascular and multifactorial diseases and employs a variety of viruses each with specific qualities making them suitable for their chosen application [1]. Gene therapy includes several different strategies the first is described as gene supplementation therapy; that is to successfully supplement defective genes that cause disease, with wild type copies delivered to target cells, as in the case of cystic fibrosis [2]. The second is cell factory gene therapy, which uses DNA or RNA as a therapeutic for expression in irrelevant cells to treat deficiencies such as diabetes [3]. Third is treatment of tumours [4] which relies on delivering the DNA or RNA normally with the purpose of killing the target cell. Killing strategies can involve expression of cytotoxic agents, enzymes capable of activating produgs to toxic metabolites (“suicide enzymes”) or immune-provoking proteins. Alternatively the use of a replication competent (or conditionally replicating) lytic viral vector can cause cell lysis. In addition there are other strategies—notably the encoding of hairpin RNA to produce small inhibitory RNA (siRNA) in situ to downregulate expression of target mRNA, encoding oligonucleotides to promote exon skipping, ribozymes and antisense strategies.
In all cases successful delivery of the gene delivery vector (which can be viral or synthetic) would be improved if the vector had selectivity for binding and entry into target cells.
Adenoviruses are widespread in nature, infecting birds, mammals and man. Belonging to the family Adenoviridae and the genus Mastadenovirus, over 50 human adenovirus serotypes have been classified within 6 subgenera (A-F), according to their hemaglutination pattern, their DNA homology and other criteria. The most prevalent serotypes are those of subgenus C (1, 2, 5 and 6). Together with some serotypes of subgenus B and E these viruses are a frequent cause of acute upper respiratory tract (URT) infections and other respiratory pathologies. In addition, Adenoviruses also cause a number of other types of infection often associated with the eye (e.g. conjunctivitis and epidemic keratoconjunctivitis), the gastrointestinal tract (e.g. gastroenteritis) or the urogenital tract (e.g. cystitis). The organ tropism is distinct for different human adenovirus subgenera. Adenoviruses have also been used therapeutically for vaccination and for gene therapy.
The adenovirus is an example of a virus typically used for gene therapy [5]. It has an icosahedral structure with 12 protruding fibre proteins, which possess a specifically folded arrangement of protein at the end called the knob domain that binds via a three-way interaction with the coxsackie adenovirus receptor (CAR) present on many cell membranes [6]. This is known to be the major route of infection, although alternative pathways involve binding of the virus to cell surface integrins and heparin sulphate proteoglycans [7]. Specific integrin mediated uptake is also known [7].
A preferred vector would combine powerful ability for transgene expression with a high level of cell or tissue specificity. This problem has been addressed previously.
For example, cell targeting has been achieved with adenovirus vectors by selective modification of the viral genome knob and fibre coding sequences to achieve expression of modified knob and fibre domains having specific interaction with unique cell surface receptors. Examples of such modifications are described in Wickham, et al (1997) J. Virol 71(11): 8221-8229 (incorporation of RGD peptides into adenoviral fibre proteins); Arnberg, et al (1997) Virology 227:239-244 (modification of adenoviral fibre genes to achieve tropism to the eye and genital tract); Harris and Lemoine (1996) TIG 12(10):400-405; Stevenson, et al (1997) J. Virol. 71(6): 4782-4790; Michael, et al (1995) Gene Therapy 2:660-668 (incorporation of gastrin releasing peptide fragment into adenovirus fibre protein); and Ohno, et al (1997) Nature Biotechnology 15:763-767 (incorporation of Protein A-IgG binding domain into Sindbis virus).
Other methods of cell specific targeting have been achieved by the conjugation of antibodies or antibody fragments to the envelope proteins (see, e.g. Michael, et al. (1993) J. Biol. Chem. 268:6866-6869, Watkins, et al. (1997) Gene Therapy 4:1004-1012; Douglas, et al. (1996) Nature Biotechnology 14: 1574-1578. Alternatively, particular moieties may be conjugated to the viral surface to achieve targeting (See, e.g. Nilson, et al. (1996) Gene Therapy 3:280-286 (conjugation of EGF to retroviral proteins). Additionally, cell specific targeting may be achieved by placing the virally encoded transgene under the control of a tissue specific promoter region allowing expression of the transgene preferentially in particular cell types.